Parasitic nematodes remain a significant public health problem in many parts of the world. Ascaris infects over 1 billion people, decreases physical and cognitive development in children, and hinders socioeconomic development in endemic areas. In order to help combat these parasitic infections, greater knowledge of parasite biology is needed. One new avenue of research yet to be explored in parasitic nematodes is small RNAs that act as "guides" in gene silencing. Small RNAs (19-32 nt) regulate mRNA translation and stability, lead to gene silencing through the formation of heterochromatin, and are involved in DNA elimination and rearrangements. Nothing is known regarding the spectrum of small RNAs present in parasitic nematodes. Ascaris represents a unique nematode system for analysis of small RNAs because 1) large numbers of synchronously developing embryos can be obtained, 2) individual tissues with discrete developmental regions such as the male and female reproductive systems can be isolated, and 3) an unusual form of DNA elimination (chromatin diminution in which 25% of the genome is eliminated) in the somatic lineages occurs during early development. We will prepare and "deep sequence" Ascaris small RNA libraries from several early developmental stages and regions of the male and female reproductive systems. Sequences from the small RNA libraries will enable us to test the following hypotheses: 1) Expression of a discrete set of small RNAs is temporally associated with chromatin diminution in early Ascaris embryos, 2) Small RNAs are differentially expressed and are temporally associated with regulation of mRNA translation and stability prior to the onset of zygotic transcription and in early development, and 3) Small RNAs are uniquely associated with the germline, gametogenesis, and organogenesis. Furthermore, we will also initiate studies to develop methods for in depth mechanistic analyses of the role of small RNAs in mRNA translation and stability in Ascaris using our cell-free and in vivo systems. Our studies will identify and characterize parasitic nematode small RNAs in specific stages and tissues and at a depth heretofore not previously done in a nematode. Overall, our analysis of small RNAs in Ascaris, a parasitic nematode with tremendous reproductive capacity, may identify novel small RNAs associated with parasitic nematodes. These studies are likely to identify new players in parasitic nematode gene expression and regulation that could be new drug targets for the development of novel therapeutics against these parasites. PUBLIC HEALTH RELEVANCE: Parasitic nematodes remain a significant public health problem in many parts of the world. Ascaris alone infects upwards of 1 billion people and hinders socioeconomic development in endemic areas. These studies are likely to identify new players in parasitic nematode gene expression and regulation that could lead to the development of novel therapeutics against parasitic nematodes.